Columns used in liquid chromatography typically comprise a tubular or cylindrical body enclosing a packed bed of porous chromatography medium through which a carrier liquid flows, with separation taking place by partitioning between the carrier liquid and solid phase of the porous medium. Cylindrical columns are generally known as ‘axial’ columns, chromatographic separation typically occurring in a vertical direction down the length of the column, while tubular columns are generally known as ‘radial’ columns with separation taking place in a radial direction as the carrier liquid flows to the centre of the cylinder.
Prior to any separation process, the bed has to be prepared by starting from the particulate medium that is to be introduced into the column. Conventional columns aimed for high efficiency separations deploy a process of bed formation, which is called ‘the packing procedure’. In high efficiency separations, a correctly packed bed is a critical factor influencing the performance of a column containing a packed bed. Typically, the packed bed is prepared by slurry packing, i.e.
consolidating a suspension of discrete particles or fibres in liquid, known as slurry that is pumped, poured, or sucked into the column. Once the predetermined volume of slurry has been delivered into the column it needs to be further consolidated and compressed.
In axial columns or in axial chromatography, the slurry can be compressed by moving a movable adapter down the longitudinal axis of the column towards the bottom of the column, normally at a constant speed. The excess liquid during this procedure is expelled at the column outlet, while the media particles are retained by means of a filter material, a so-called ‘bed support’ or ‘frit’, with pores too small to allow the media particles to pass though. The packing process is complete once the packed bed has been compressed by the optimum degree of compression.
Another approach for column slurry packing used both in axial and radial columns is the flow packing method, where compression of the porous structure is primarily achieved by applying a high flow rate over the column, thereby forming a porous structure starting at the outlet bed support. The resulting drag force on the particles in the porous structure causes eventually a pressure drop and a compression of the bed. The compressed bed is finally confined by bringing the adapter into position. Presently, only flow packing methods are known for packing radial columns.
The efficiency of subsequent chromatographic separation relies strongly on 1) the liquid distribution and collection system at the fluid inlet and outlet of the packed bed, 2) the special orientation (also know as the packing geometry) of the media particles in the packed bed, and 3) the compression of the packed bed. If the compression of the packed bed is too low then chromatographic separations performed on that bed suffer from “tailing” and, generally, such insufficiently compressed beds are unstable. If the compression of the packed bed is too high then chromatographic separations performed by the bed suffer from “leading” and such over-compressed beds can affect throughput and binding capacity, and, in general, give much higher operating pressures. If the compression is optimum, then the separation peaks formed during use exhibit much less leading or tailing and are substantially symmetrical. The optimum degree of compression is also crucial for achieving good long-term stability of the porous structure, thereby securing optimal performance throughout a number of process cycles. The optimum degree of compression required for a column is determined experimentally for each column size (width or diameter), bed height, and media type.
An alternative packing method used for axial columns is called “dry packing”, where the column is filled with dry particles of the porous medium and liquid is introduced in the column afterwards. This has advantages in prepacked columns that can be delivered dry to the customer without having to add any preservatives to the packing liquid and minimizing weight during transport. Dry packing is typically used for silica media aimed at separation of small molecules, as described e.g. by G Guiochon J Chromatogr A 704 (1995) 247-268, although fairly poor column efficiencies are obtained. For swellable chromatography media, such as dextran or agarose-based media commonly used in separation of biomolecules, dry packing has however been avoided due to a perception that the swelling of the particles will cause poor performance of the packed bed.
The above described background for preparation of packed beds adapted for high efficiency chromatographic separations applies equally to packed beds or fixed beds aimed for achieving bio-reactions such as enzymatic conversions, the treatment of cells or the growth and cultivation of cells adherent to the packed bed with high efficiency.
The columns and packed beds described above may also be used for stabilisation of substances and in particular biomolecules such as proteins, antibodies and cells. This stabilistation may improve and facilitate storage and transport of the molecules, for example. The binding of substances, particularly biomolecules, to chromatography media may preserve activity and stability of the substance more than is achievable with other concentration methods such as filtration, precipitation or freeze-drying.